Callus culture and microgravity application
H. niger seeds were collected from Maragheh, Azerbaijan Province of Iran. Seeds were sterilized in a 10 % (v/v) NaOCl solution for 10 min and then, washed three times with sterile distilled water. Then, sterilized seeds were placed in 70% alcohol for 1 min and 10% sulfuric acid for 7 min, followed by three times of washing with distilled water. Next, the disinfected seeds were placed in half strength (½) Murashige and Skoog (MS) basal medium(Murashige 1962), supplemented with 7% agar and 3% sucrose under dark condition, at 25 ± 2 and pH 5.7. For callus induction, hypocotyl segments (0.4-0.5 cm) of 10 days old seedlings were transferred into solid MS medium, supplemented with 1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L−1 benzyl amino purine (BAP) hormones and incubated in the darkness for three weeks. After that, the calli were sub-cultured using the same MS solid medium. For simulated microgravity exposure, calli were placed at the center of Petri dish with a 2 cm rotational radius and located on a developed two-dimensional clinostat turning at the rate of 2 rpm, for 0 (control), 3, 7 and 10 days. The centrifugal force was evaluated from zero on the center to 2.24 × 10–5 g on the edge of the callus ring. Finally, 3-5 calli per treatment were used for the evaluation of H. niger cell growth and biochemical analysis.
Hydrogen peroxidase (H2O2) measurement
To evaluate H2O2 content of the H. niger, we homogenized microgravity-treated samples in 1 ml solution containing 0.25 Trichloroacetic acid (TCA) (0.1% (w:v)), 0.5 ml KI (1M) and 0.25 ml potassium phosphate buffer (10 mM), at 4 for 10 min. Simultaneously, control samples were prepared with H2O instead of KI for tissue coloration background. Homogenized samples were centrifuged at 12000 ×g, 4 for 15 min and then, 200 µl supernatant of each sample was placed in a UV-microplate well, followed by a 20 min incubation at room temperature. Finally, the absorbance of all samples was read at 350 nm, using Shimadzu UV-1800 spectrophotometer. The standard curve was also obtained with H2O2 standard solutions, prepared in 0.1% TCA (Velikova et al. 2000)
For the estimation of lipid peroxidation, we measured the malondialdehyde (MDA) content of H. niger calli, based on the Heath and Packer modified protocol (Heath and Packer 1968). To do this, 0.3 g fresh callus samples were homogenized in 25% Thiobarbituric acid (TBA) reagent and 10 % TCA. The reaction mixture was boiled in a water bath, at 95°C for 20 min. After incubation, the mixture was quickly cooled on ice and centrifuged at 10000 ×g, 4 (Sigma Centrifuge 1-16K, German). Measurement of the supernatant absorbance was performed at 532 and 600 nm, using a UV–visible spectrophotometer (UV-160, Shimadzu, Tokyo, Japan). MDA content was determined by the extinction coefficient of 155 mM−1 cm−1.
Total protein content and enzymatic activity
Following the extraction of 0.2 g fresh H. niger calli with the use of 0.1 M sodium phosphate extraction buffer, at 4 and pH 6.8 (EMSURE®, MerckKGaA), samples were centrifuged at 10,000 rpm for 15 min, at 4 . The obtained supernatant was stored at −70 and used for the measurement of the protein and enzyme activity. Soluble protein content was evaluated through Bradford method (Bradford 1976). The enzymatic extract (100 µL) was added to the reaction mixture containing 25 mg Coomassie Brilliant Blue, 12.5 mL ethanol 96%, and 25 mL phosphoric acid 85%. After 10 min of incubation, the absorbance was measured at 595 nm (Shimadzu UV-1800 spectrophotometer).
Catalase activity was evaluated according to the proposed method of Dhindsa, et al (Dhindsa et al. 1981). The enzymatic extract (100 µL) was added to the reaction mixture containing 0.2 mL H2O2 3% and 2.8 mL phosphate buffer. Subsequently, the absorbance was measured at 240 nm through Shimadzu UV-1800 spectrophotometer.
The evaluation of peroxidase activity was performed through the method of Abeles and Biles (Abeles and Biles 1991). The enzymatic extract (100 μL) was added to the reaction mixture containing 0.2 mL H2O2 3%, 2 mL acetate buffer 0.2 M at the pH of 4.8 and 0.1 mL benzidine at the concentration of 20 mM. The absorbance was recorded at the wavelength of 530 nm (Shimadzu UV-1800 spectrophotometer).
Determination of Proline and sugar content
The analysis of proline content was conducted according to the acid ninhydrine procedure of Bates et al (Bates et al. 1973). The callus samples (0.2 g) were pulverized with 4 ml sulfosalicylic acid 3% and clarified through centrifugation. Then, 2 ml of the obtained supernatant was mixed with the same volume (2 ml) of acid ninhydrin and acetic acid, followed by incubation in the oven, at 100 for 1 h. The reaction was stopped by putting the mixture on the ice. In order to separate the reaction mixture, 4 ml toluene was added and vortexed for 15-20 s. The absorbance of the samples was measured at 520 nm, using Shimadzu UV-1800 spectrophotometer.
To evaluate the content of soluble sugars, calli samples (0.2 g) were dissolved in 10 mL (80% v/v) ethanol and centrifuged at 6000 ×g, for 10 min. Total soluble sugars were determined through preparing the mixture of 1 mL supernatant and 3 mL anthrone reagent [200 mg anthrone in 100 ml H2SO4 (70% v/v)] (EMSURE®, MerckKGaA), and the following incubation in the boiling water bath for 10 min. Then, the absorbance was recorded at the wavelength of 620 nm, using Shimadzu UV-1800 spectrophotometer. The total soluble sugar content was also determined through a calibration curve of Glucose, being expressed as gram of tissue weight equivalent.
Determination of Phenyl-alanine amonialyase activity
To measure the activity of PAL enzyme, we prepared a stock solution of 0.006 mM (5 ml) phenyl alanine and 0.5 M (pH 8; 120 ml) Tris-HCL buffer as the reaction mixture. Then, 0.4 ml enzymatic extraction was added to 6 ml reaction mixture. Control samples consisted of 6 ml reaction mixture and 0.4 ml water, instead of enzymatic extraction. After stirring and incubation at 37 for 1 h, 0.1 ml HCL was added to stop the reaction. The absorbance was recorded at the wavelength of 290 nm (Shimadzu UV-1800 spectrophotometer)(Beaudoin-Eagan and Thorpe 1985).
Determination of Atropine and Scopolamine by HPLC
Atropine and Scopolamine were extracted from the callus samples (0.2 g of which were dried samples) and then underwent an overnight incubation in the mixture of 28% ethanol and NH4OH at the ratio of 9:1, respectively. Then, the extracted samples were incubated in the ultrasonic bath for 30 min and centrifuged at 1,500 rpm, for 3 min. The sediment was dissolved in 1.5 mL HCl 0.1 N and the prepared acidic aqueous solution was filtered to produce alkaline with diluted KOH (final pH 8-9). Next, 6 ml chloroform was added and after a vigorous shake, the tube was centrifuged at 1,500 rpm, for 2 min. The lower layer which contained alkaloids underwent a double precise pipetting with 6 mL chloroform and subsequently was evaporated at 40 until dryness. The dry sediment was dissolved in 99% methanol to determine the Atropine and Scopolamine content through High Performance Liquid Chromatography (HPLC, Cecil Company, England), using an ODS (C18) column (25 cm × 4.6 mm, partial size 5 µm). The mobile phase contained 450 mM Acetonic-K2HPO pH 3 by phosphoric acid; flow rate: 1.5 mL/min; UV K2501 detector at 280 nm, and was analyzed through an external standard (Kamada et al. 1986).
Gene expression analysis
H. niger samples (50 mg) were homogenized in liquid nitrogen and RNA was isolated using DENAzist Column RNA Isolation Kit (DENAzist Asia Co., Mashhad, Iran). In order to avoid any DNA contamination, 3 µg of total RNA was treated with DNase I kit (EN0521, fermentase) and then, the purity of the extracted RNA was checked through NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) and 1% agarose gel electrophoresis. After reverse transcription of RNA into cDNA (SMOBIO cDNA synthesis kit, Taiwan), the relative expression of target genes (Table 1) was analyzed by Applied Biosystems Step One real-time PCR systems in triplicate reactions per samples, using SYBR Green Master Mix (Amplicon RealQ Plus 2x, High ROX™, Denmark) with an initial denaturation step (5 min at 95 ) and the subsequent 40 cycles of 15 s at 94˚C, 25 s at 57 and 20 s at 72 . The EF1α was used as the internal reference gene. Relative expression of treatments were calibrated with control samples and calculated by 2-ΔΔCt (Schmittgen and Livak 2008).
All data were expressed as mean ± SD of at least three replications. Data analysis was done by one-way analysis of variance (ANOVA), with the Brown-Forsythe test and the use of GraphPad Prism 9.0 (San Diego, USA). P values lower that 0.05 were considered as statistically significant.